(Taken from application) The goal of this project is to carry out studies that will evaluate the hypothesis that abnormalities in signal transduction mechanisms, especially those involving metabolism of phospholipids and their component fatty acids are critically involved in the complex events comprising the pathogenesis of experimental diabetic neuropathy. The mechanism by which aldose reductase inhibitors and antioxidants, especially RRR -alpha-tocopherol (Vitamin E), exert their beneficial effects on diabetic nerve will also be studied.
The specific aims of this project are as follows: 1.Investigation of the possible relationship between arachidonic acid (AA) metabolism, the polyol pathway and antioxidant treatment in human primary and tumor-derived (NF1T) Schwann cell lines, as well as in primary neonatal rat and human fetal Schwann cells by examining the effects of aldose reductase and sorbitol dehydrogenase inhibitors as well as Vit E and N-acetylcysteine, on arachidonyl containing molecular species (ACMS) levels and arachidonate turnover. 2. Exploration of the mechanism underlying reduced ACMS levels and altered AA turnover in NF1T cells grown in elevated glucose by : a) measurement of free fatty acid and acyl CoA levels; b) assay of delta-6 desaturase activity; c) assay of PLA2 activity; d) determination of NADP+/NADPH ratio e) identification of AA metabolites i.e. prostaglandins and HETEs, released from the cells and the ability of the cells to synthesize these compounds. 3. Complementary experiments will be performed by feeding normal and streptozotocin -induced diabetic rats diets supplemented with : a) an aldose reductase inhibitor; b) Vit E ; c) both agents together ; d) a Vit E deficient diet. The effects of these dietary regimens on nerve ACMS, DAG levels and PKC activity will be assessed. These analyses will be correlated with morphological examination and nerve conduction velocity measurements. In addition it will determine whether ACMS levels are reduced in normal and transgenic mice that express human aldose reductase. 4. Investigations on the expression of several antioxidant enzymes in nerve and dorsal root ganglia from normal and STZ-induced diabetic rats with respect to their mRNA levels and protein, as well as enzyme activities. The enzymes will include Mn superoxide dismutase, glutathione peroxidase and catalase. The impact of Vit E supplementation and imposition of a Vit E deficiency on expression and activity of the enzymes will also be examined.
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